The invention herein disclosed is directed to immunoassays involving liposomes or other microspheres.
The basis for the invention is a modification of the Liposome Immune Lysis Assay (LILA), whose theoretical principle is the following:
1. Prior Art Method
Antibody-dependent cytotoxicity is a process mediated by immunoglobulins and a number of host proteins termed the xe2x80x9ccomplement systemxe2x80x9d. When antibodies recognize and bind their target, they will suffer conformational changes that activate the complement system, which will eventually result in the formation of the xe2x80x9cmembrane attack complexxe2x80x9d on the surface of the foreign substance or cell. It is the membrane attack complex that perforates the cytoplasmic membrane and induces membrane channels that result in the lysis of the cell targeted by the antibodies. This perforation of membranes is utilized to evaluate the presence of circulating antibodies. Instead of using cells or organisms, the LILA assay uses liposomes, which are small spheres composed of lipids. These liposomes have the property of allowing a substance placed in their interior to remain hidden from the environment as long as the lipid capsule or liposome remains intact. When the capsule is destroyed or punctured, its contents are released. The prior art discloses liposomes that contain a substance which when released from the liposome will change color when contacting an appropriate substrate present in the surrounding environment. Further, liposomes may be sensitized with an antigen on their surface (i.e., cardiolipin). When the sensitized liposomes are diluted with serum of a patient with antibodies to said antigen, in this case against the syphilis antigen, i.e., cardiolipin, the antibodies recognize and attach to the surface of the sensitized liposomes, thus activating the complement present in the serum, which in turn activates the membrane attack complex. Once the complement system is activated and starts puncturing the liposome surface, the substance contained in the interior of the liposome (e.g., an enzyme that may change the color of a substrate) leaks into the surrounding solution which contains the appropriate substrate for the enzyme. The color of the substrate changes due to the enzyme acting thereon. This color change can then be measured in a spectrophotometer or a device used for reading ELISA assays. This is an ingenious assay, but it does not provide advantage over other methods such as solid phase immunoassays such as the ELISA, which are used to measure the same substance.
2. Prior Art
Tomioka et al in the Journal of Immunological Methods, Vol. 176 (1994) pages 1-7, teach immunoassay methods using liposomes using a model lipid of archaebacteria, namely, 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphlocholine. The advantage of this model lipid is that it could be used along with other lipids to form stable liposomes, as explained in the article.
Cole in U.S. Pat. No. 4,483,921 teaches immunoassays involving liposomes. In that assay after recognition between antigen and antibody, the liposome ruptures releasing an enzyme which is detected indicating a positive results.
Wagner et al U.S. Pat. No. 4,978,625 uses liposomes in immunoassay, however, the assay does not depend upon the rupture of the liposome.
Gibbons et al (U.S. Pat. No. 5,068,198) describes preparing liposomes to be used in immunoassays. The substance in the liposome is released, for example, by freezing and thawing, sonication or osmotic shock.
Hosoda et al (U.S. Pat. No. 5,173,406) discloses a liposome immunoassay in which complement is involved in causing the rupture of liposome. A kit for carrying out the assay is taught. Various markers used in the assay are disclosed. Hosoda et al also disclose the use of polyclonal, as well as monoclonal antibodies.
Kida et al (U.S. Pat. No. 5,221,613) disclose an immunoassay process using liposomes. The assay encapsulates an antibody in the liposome.
Malick et al (U.S. Pat. No. 5,620,903) teaches liposome assays. Water-insoluble dyes are used as the marker with water-soluble dyes added as an option.
These prior art patents show liposomes used to encompass reagents in various embodiments, however, none of the prior art reacts guaiac disposed in a liposome with blood hemoglobin.
A main object of this invention is to produce a method that will allow for the accurate and fast detection of a large number of human diseases using a very economical and simple one-step method.
A further object is to use sensitized liposomes or microspheres containing a reagent to detect a blood component.
A specific object of this invention is to use a sensitized liposome containing guaiac to react with hemoglobin in the event a specific antibody is detected.
The present disclosure describes an homogeneous immunoassay designed to detect the presence of specific antibodies in whole blood. Such antibodies react with an antigen present on the surface of microcapsules such as liposomes, which enclose a reporter molecule. Subsequent to the antibody-antigen reaction, the microcapsules lyse by the action of the complement system, liberating their content which then reacts with a substance or substances present in whole blood, producing a change in color which can be visible to the naked eye.
The herein disclosed invention has a main object the rapid detection of an antigen or antibody in the blood.
Another object is to produce an immunotest which can readily detect a disease state in the body by testing blood.
The invention contemplates a test kit for immunoassay of a blood sample comprising in effective amounts:
a) an antigen or antibody sensitized liposome containing guaiac therein,
b) complement and
c) hydrogen peroxide
The invention also contemplates a method for detecting either an antigen or antibody in a hemoglobin-containing blood sample comprising
a) providing a container containing a sensitized liposome surrounding guaiac therein,
b) said container also being provided with complement and then
c) adding a hemoglobin containing blood sample to be tested for a specific antigen or antibody such that if there is a reaction between the antigen or antibody on the sensitized liposome, with its specific binding partner, the liposome will rupture, releasing the guaiac to react with the hemoglobin contained in the blood to produce a blue/brown-black color indicating a positive result; and in the event there is no reaction between the specific binding partners the liposome will remain intact and the blood sample will retain its original coloration indicating a negative result. More specifically the liposome can be sensitized to syphilis.
In a broad aspect, the described invention envisions in combination, a composition to be tested comprising a blood sample and a sensitized liposome containing a reagent which will react with a blood component.
In a broad aspect, the invention involves a method for detecting either an antigen or antibody in a blood sample comprising combining said blood sample with a sensitized liposome containing a chemical agent which will react with a blood component once a specific binding partner for the sensitized liposome causes the liposome to rupture.
This invention contemplates in combination, a composition to be tested comprising a blood sample and a sensitized microsphere containing a reagent which will react with a blood component, once the microsphere is ruptured.
One aspect of this invention may be viewed as a method for detecting either an antigen or antibody in a blood sample comprising combining said blood with sensitized microspheres containing a chemical agent which will react with a blood component once a specific binding partner for the sensitized liposome causes the liposome to rupture.
Gibbons et al (U.S. Pat. No. 5,068,198) discloses materials for reversibly surrounding the reagents of this invention and are materials that are capable of temporarily confining the reagent until a reaction is effectuated and then releasing the confined reagent. Illustrative materials include lipid bilayers such as liposomes, artificial cells, vesicles, natural cell membranes such as red blood cells ghosts; gels such as gelatin and agarose; polymerized beads and the like. Applicants"" invention visualizes employing these microspheres or microcapsules in their invention. Applicants also refer to U.S. Pat. No. 5,128,241 as emplifying xe2x80x9cerythroctye ghost membranexe2x80x9d as an operative microcapsule to use in their invention. Note is also taken of Malick et al (U.S. Pat. No. 5,620,903) who disclose the use of silicon for forming microspheres. The disclosures of these patents are incorporated by reference into the disclosure of this application.